5-Lipoxygenase Interacts with Coactosin-like Protein

Abstract

We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca 2؉ -independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP⅐5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics. 5-Lipoxygenase (5LO)1 is of central importance in cellular leukotriene (LT) synthesis. This enzyme converts arachidonic acid released from the membranes by the cytosolic phospholipase A 2 into 5(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and subsequently into the epoxide intermediate LTA 4 (1). LTA 4 is further metabolized into LTB 4 by the LTA 4 hydrolase or into LTC 4 through the action of the LTC 4 synthase. LTC 4 is then sequentially degraded into LTD 4 and LTE 4 .Whereas LTB 4 exerts potent stimulatory effects on various leukocyte functions, including chemotaxis, adhesion, degranulation, and aggregation, the cysteinyl-LTs (LTC 4 , LTD 4 , and LTE 4 ) are known to contract airway smooth muscle, increase vascular permeability, and promote mucus secretion (2). 5LO and LTs are, therefore, key components involved in inflammatory disorders, including arthritis, asthma, and allergic reactions.Recently, novel modulatory mechanisms determining cellular 5LO activity were identified. 5LO is phosphorylated by p38 mitogen-activated protein kinase-activated protein (MAPKAP) kinases prepared from stimulated myeloid cells (3). In addition, Mg 2ϩ increases 5LO activity in vitro (4). Furthermore, a stimulatory Ca 2ϩ binding site has been localized in the N-terminal domain of 5LO, that may function as a C2 domain in the calcium regulation of 5LO catalytic activity (5). C2 domains have also been shown to mediate protein-protein interactions (6).Additional lines of evidence indicate that cellular 5LO activity and distribution is regulated by interaction with other proteins. For example, the subcellular distribution of 5LO differs among cell types and changes in response to various stimuli. In particular...

Roles of coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP) in cellular leukotriene biosynthesis

“…S3). We previously reported that binding of the purified proteins in vitro occurred in the absence of Ca 2+ , and that epitope-tagged constructs (e.g., FLAG-5LO, Myc-CLP) could be efficiently coimmunoprecipitated after overexpression in HEK 293 cells (no ionophore stimulation) (15). Those findings suggested a static association between CLP and 5LO, whereas the ionophore-inducible binding seen in the present study indicates a different situation for the native proteins in leukocytes with high capacity for LT biosynthesis.…”

“…Human CLP was first identified as a 5LO-binding protein in yeast two-hybrid screening with 5LO as bait (14). Native PAGE and chemical cross-linking experiments showed that in vitro 5LO binds CLP with 1:1 molar stoichiometry (15). A member of the ADF/cofilin group of actin-binding proteins, CLP also binds F-actin (16).…”

Analysis of 5‐lipoxygenase phosphorylation on molecular level by MALDI‐MS

“…Therefore, the most effective approach for efficient 5-LO purification was the overexpression of tagged 5-LO. For this purpose, we used a gene encoding FLAG-tagged 5-LO, for which it has been demonstrated that the activity of the resulting enzyme is similar to non-tagged 5-LO [30]. From the stably transduced MM6 cells, anti-FLAG tag IP could efficiently purify FLAG-5-LO from the total cell lysates, independent of cell stimulation with known activators of the leukotriene pathway (priming with LPS with/without subsequent stimulation with the bacterial peptide fMLP or stimulation by 10 lM arachidonic acid and 5 lM Ca 2+ -ionophore A23187, Fig.…”

“…RPMI 1640 medium supplemented with 2 mM L-glutamine, penicillin/streptomycin solution, sodium pyruvate for cell culture and Dynabeads Ò RPC 18 were from Life Technologies GmbH (Darmstadt, Germany). Codon-optimized sequence expressing human 5-LO protein with an N-terminal FLAGtag, the latter according to Provost et al [30], was created by GENEART AG Life Technologies GmbH (Darmstadt, Germany). Infrared dye-conjugated antibodies (IRDye) and Odyssey Blocking Buffer were purchased from LI-COR Biosciences (Bad Homburg, Germany).…”

Solution structure of coactosin reveals structural homology to ADF/cofilin family proteins

“…Dictyostelium discoideum coactosin was also reported to show a weak uncapping activity [10]. In addition, Provost et al [11] demonstrated that mouse coactosin binds 5-lipoxygenase, an enzyme involved in leukotriene biosynthesis, although the biological role of this interaction is unknown. First cell biological studies suggest that coactosin colocalizes with the actin cytoskeleton in cultured cells [8,9].…”